2020 RIBEIRO Short Duration B Alanine Supplementation Did Not Prevent The Detrimental Effects of An Intense Preparatory Period On Exercise Capacity

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ORIGINAL RESEARCH

published: 21 April 2020


doi: 10.3389/fnut.2020.00043

Short-Duration Beta-Alanine
Supplementation Did Not Prevent the
Detrimental Effects of an Intense
Preparatory Period on Exercise
Capacity in Top-Level Female
Footballers
Rafael Ribeiro 1 , Breno Duarte 1 , André Guedes da Silva 1 , Guilherme Passos Ramos 2 ,
Andreia Rossi Picanço 2 , Eduardo Macedo Penna 3 , Victor Coswig 3 , Matheus Barbalho 4 ,
Paulo Gentil 4 , Bruno Gualano 1 and Bryan Saunders 1,5*
1
Applied Physiology and Nutrition Research Group, School of Physical Education and Sport, Rheumatology Division,
Faculdade de Medicina FMUSP, Universidade de São Paulo, São Paulo, Brazil, 2 Confederação Brasileira de Futebol, Rio de
Janeiro, Brazil, 3 Physical Education, Federal University of Pará – Campus Castanhal, Castanhal, Brazil, 4 Physical Education
and Dance Institute, Federal University of Goiás, Goiânia, Brazil, 5 Institute of Orthopaedics and Traumatology, Faculty of
Edited by: Medicine FMUSP, University of São Paulo, São Paulo, Brazil
David Christopher Nieman,
Appalachian State University,
United States Purpose: High-intensity activity is an important aspect of football performance during
Reviewed by: competitive match play. The aim of this study was to investigate the effect of beta-alanine
David Hooper,
supplementation throughout a short-duration intense football-specific training period
Jacksonville University, United States
Sophie C. Killer, prior to an international competition on measures of high-intensity running performance.
English Institute of Sport,
United Kingdom
Methods: Twenty-four elite international U20 female footballers (age 18 ± 1 y, height
*Correspondence:
1.67 ± 0.07 m, body mass 62.7 ± 7.4 kg) volunteered to perform the YoYo Intermittent
Bryan Saunders Recovery Test Level 1 (YoYo IR1), the Running Anaerobic Sprint Test (RAST) and a 20-m
drbryansaunders@outlook.com
maximal sprint test on two separate occasions, separated by 3 weeks of training and
Specialty section:
supplementation. Participants were randomly assigned to receive either 6.4 g·day−1
This article was submitted to sustained-release beta-alanine (BA, N = 12) or an equivalent dose of maltodextrin
Sport and Exercise Nutrition, (placebo, PL, N = 12) throughout a 3-week standardized training camp.
a section of the journal
Frontiers in Nutrition Results: There was a main effect of group (P = 0.05) and time (P = 0.004) on YoYo
Received: 13 January 2020 IR1; overall values were lower in PL and distance covered was lower post- vs. pre-
Accepted: 20 March 2020
supplementation. There was no group × time interaction (P = 0.07). There was an effect
Published: 21 April 2020
of sprint number for RAST, but no further main effects and there were no effect for the
Citation:
Ribeiro R, Duarte B, Guedes da 20-m sprint.
Silva A, Ramos GP, Rossi Picanço A,
Penna EM, Coswig V, Barbalho M,
Conclusions: Top-level female footballers involved in this intense 3-week training period
Gentil P, Gualano B and Saunders B prior to a competition worsened their high-intensity intermittent exercise capacity, and
(2020) Short-Duration Beta-Alanine
this negative result was not attenuated by a short-duration BA supplementation protocol
Supplementation Did Not Prevent the
Detrimental Effects of an Intense throughout the same period. Further work is necessary to elucidate whether adapted
Preparatory Period on Exercise training protocols and BA dosing regimens could lead to better results.
Capacity in Top-Level Female
Footballers. Front. Nutr. 7:43. Keywords: football training, nutritional supplementation, YoYo intermittent recovery test, repeated sprints,
doi: 10.3389/fnut.2020.00043 competition, fatigue, elite

Frontiers in Nutrition | www.frontiersin.org 1 April 2020 | Volume 7 | Article 43


Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers

INTRODUCTION considered an effective ergogenic aid by the International


Olympic Committee (18), although the effects of beta-alanine
Football, also commonly termed as soccer, is the world’s most on football-specific protocols is unclear and contradictory.
popular sport, practiced by men and women all around the Beta-alanine has previously improved YoYo IR2 performance
world (1). The women’s game has seen a stark increase in in amateur male footballers throughout a competitive season
both popularity and professionalism over the past decade, (19), although YoYo IR1 was not improved in young elite
with research into the physiological demands of the women’s male basketball players (20). Evidence to support beta-alanine
game following a similar rise in popularity (2). The general supplementation during shorter-duration repeated sprints is
characteristics of women’s football demonstrate that match- distinctly lacking (21–23) although supplementation alongside
play is predominantly performed at low-intensity activities plyometric training did lead to greater improvements in RAST
interspersed by numerous high-intensity actions throughout than training alone in female soccer players (11). This suggests
(2, 3). Key moments that can affect the outcome of a game that the combination of training and beta-alanine may be
generally occur at high-intensities. High-intensity efforts are additive, something previously demonstrated with cycling sprint
reduced during various phases of international matches and vary training (24), although no study to date has investigated the
according to position (4) and top-level women players have combined effect of a football-specific training period alongside
been shown to perform more high-intensity running and sprints beta-alanine supplementation on football-specific performance
during games than their less successful counterparts (5). High- in females.
intensity activity, therefore, appears to be an important aspect of The aim of this study was to investigate the effect of beta-
football performance during competitive match play. alanine supplementation throughout a short-duration intense
Several field tests are employed to evaluate the training football-specific training period prior to an international
status of football players and are commonly used to predict competition on measures of high-intensity running performance.
match performance and determine the effectiveness of a training We hypothesised that supplementation would lead to greater
intervention. The YoYo Intermittent Recovery Tests (Level 1 improvements in exercise measures than any seen with
[YoYo IR1] and 2 [YoYo IR2]) evaluate an individual’s capacity training alone.
to repeatedly perform and recover from intense exercise bouts,
and is applicable to team sports players due to the specificity of
METHODS
the exercise undertaken (6). The Yo-Yo IR1 and IR2 have been
shown to correlate to various variables of match performance Participants
and can be used as an indicator of the physical performance Twenty-four elite international under-20 (U20) female
of elite female players throughout competitive matches (7, 8), footballers (age 18 ± 1 y, height 1.67 ± 0.07 m, body mass
making them appropriate models to examine the effect of any 62.7 ± 7.4 kg) from different clubs competing in the elite
intervention designed to manipulate changes in performance divisions of the Brazilian football pyramid that were part of the
during team sports. In addition to differentiating between playing national Brazilian team preparing for the South American U20
standard, these tests can be used to monitor training adaptations, Women’s Championship, volunteered for the study and were
seasonal variation and determine differences between playing randomly assigned to receive either beta-alanine (BA, N = 12)
position [for review, see (9)]. The running anaerobic sprint test or placebo (PL, N = 12). Subjects had not taken any creatine
(RAST) is another protocol that has been shown to be reliable supplement in the 3 months prior to the study and had not taken
and valid to assess anaerobic power and is a good predictor of BA for at least 6 months. None of the subjects were vegetarian
short-distance running performances (10) while the 20-m sprint and, therefore, would have encountered small amounts of
test is a commonly used measure to assess team sport players (11). beta-alanine in their diet from the hydrolysis of carnosine and
Any changes in these performance measures may be reflective of its methyl derivatives in meat. The study was approved by the
an enhanced capacity to improve in-match performance and thus institution’s Ethical Advisory Committee.
are useful tools to determine the efficacy of any intervention.
The preparation period prior to an international competition Experimental Design
is a delicate one in which fitness training must ensure a All athletes in this study routinely performed the exercise
maintenance and rebuilding process following an intense season protocols as part of standard fitness testing throughout their
to ensure peak condition for the subsequent intense period respective seasons. Participants performed the YoYo IR1, RAST,
of matches (12). During this phase there may also be an and 20-m sprint test on two separate occasions, separated by
additional focus on technical and tactical preparation as opposed 3 weeks of training and supplementation. The exercise tests
to intense physical conditioning (13).This is commonly referred were performed in a standardized order: Sprint Test and the
to as the taper, which involves reducing the training loads YoYo IR1 in the morning and the RAST test in the evening.
from a previously intense program to optimise recovery and All players performed the same 3-week standardized training
maximise performance (14, 15). Athletes commonly employ program, which consisted of 1 to 2 training sessions per day and
supplementation methods to enhance any adaptations from received 5 meals per day at standardized timepoints. Sleeping
training routines. Beta-alanine is an amino acid that is ingested and waking times during the training period were controlled and
over several weeks to increase muscle carnosine content (16) identical for all athletes. Training and diet, including caffeine
and improve exercise capacity and performance (17). It is consumption, in the 24 h period prior to the first main exercise

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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers

session were standardized and the athletes repeated this prior to


the second main session.
Supplement group allocation was conducted in blocks with
groups being equalized according to performance in the
YoYo IR1. Throughout the same 3-week period, participants
were supplemented with either 6.4 g·day−1 of beta-alanine
(CarnoSynTM , NAI, USA) or placebo (maltodextrin; NAI, USA)
in sustained-release tablets. The dosing regimen consisted of
two 800 mg BA or PL tablets ingested four times per day (7
AM/12 PM/5 PM/10 PM). Participants ingested the supplements
alongside their standardized meals and a final dose before
bed, ensuring all had 100% compliance to the supplementation
regimen. No participant in either group reported any symptoms
of paraesthesia throughout supplementation.

Experimental Procedures
YoYo Intermittent Recovery Test – Level 1
The YoYo IR1 consists of repeated 2 × 20 m runs between FIGURE 1 | Distance covered during the YoYo IR 1 test in the beta-alanine
markers at progressively increasing speeds dictated by an audio (BA) and placebo (PL) groups pre- and post-supplementation. *P = 0.004.
signal. At the end of each 2 × 20 m bout, individuals performed Effect of time.
10 s of active recovery between consisting of a 10 m (2 × 5 m)
walk. The test ended if the player failed to reach the finish line
within the given time frame on two consecutive occasions or if adjustments were performed when a significant F value was
the player felt unable to continue (volitional exhaustion). The obtained, and the significance level was set a priori at P ≤ 0.05.
total distance covered (m) during the test was recorded as the Individual responses for the YoYo IR1 were calculated according
outcome measure. to time-to-completion using the spreadsheet of Swinton et al. (25)
using 90% confidence intervals, a typical error calculated from
Running Anaerobic Sprint Test (RAST) and 20-m reproducibility data (26) and a smallest worthwhile change of 0.2
sprint × the standard deviation of the control session (27). Due to issues
The RAST consisted of seven 20-m maximal sprints with a unrelated to the intervention (due to minor illness or injury, the
passive 10 s recovery period between each sprint; the start of each coaches instructed the athletes not to complete all protocols as
sprint was indicated by a beep from the photocell equipment a precautionary measure), complete data for the YoYo IR1 was
(CEFISE standard photocells, Brazil) which measured run time obtained for 20 athletes (BA = 10, PL = 10) and 22 athletes
for every individual sprint. The photocells were connected to completed the 20-m sprint (BA = 11, PL = 11); all athletes
a computer with specific software (CEFISE, Brasil) for speed completed the RAST pre- and post-supplementation.
analysis. Outcome measures were sprint time of each sprint
(s), total sprint time (s), mean, maximum and minimum
power output (W) [calculated as Power = (Body Mass × RESULTS
Distance2 )/Time3 ] and fatigue index (FI, %) [calculated as FI YoYo IR1
= (peak power - minimum power/peak power) × 100)] (10). YoYo performance was not significantly different between groups
Participants also performed 3 separate attempts of a maximum at baseline (BA: 644 ± 114 m, PL: 513 ± 125 m; P = 0.07),
20 m sprint test, with 5 min passive recovery between efforts. To although this almost reached statistical significance. This might
start the sprint, the volunteer was positioned 1 m behind the be due to missing data (2 individuals from BA and 2 from
first photocell to prevent premature activation of the timer. The PL). There was a main effect of group (P = 0.046), with lower
timing of the start of each maximal sprint was determined by overall values in the PL vs. BA group, and time (P = 0.004);
the athlete. distance covered was lower post- versus per-supplementation
(−7.4 ± 14.4%). The group × time interaction did not reach
Data Analysis statistical significance (P = 0.07; Figure 1). Individual data
Data were analysed using the SAS statistical package (SAS R
analysis revealed that no individuals in either group improved
University Edition, SAS Institute Inc., USA), and are presented performance above the smallest worthwhile change during
as mean ± 1SD unless stated. Exercise data were analysed using the YoYo IR1, although two athletes in BA and one in PL
mixed model analysis with individuals assumed as a random worsened performance.
factor and supplementation (2 levels; BA and PL) and time (2
levels; Day 0 and 20) assumed as fixed factors. Repeated sprints RAST and 20 m Sprint
during the RAST were analysed using mixed model analysis with There was no effect of group (P = 0.67) or time (P = 0.45) for
individuals assumed as a random factor and supplementation sprint times during the RAST, but there was an effect of sprint
(2 levels; BA and PL), time (2 levels; Day 0 and 20) and sprint number (P < 0.0001), reflecting an increase in time to complete
number (7 levels; 0-7) assumed as fixed factors. Tukey–Kramer each sprint with increasing sprint number (Table 1). There were

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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers

TABLE 1 | Sprint times (s) during the RAST for the beta-alanine (BA) and placebo
(PL) groups pre- and post-supplementation.

BA PL

Pre Post Pre Post

Sprint 1 (s) 3.6 ± 0.1 3.5 ± 0.1 3.5 ± 0.1 3.6 ± 0.1
Sprint 2 (s) 3.6 ± 0.2 3.6 ± 0.1 3.6 ± 0.1 3.6 ± 0.1
Sprint 3 (s) 3.7 ± 0.1* 3.7 ± 0.2* 3.6 ± 0.1* 3.7 ± 0.2*
Sprint 4 (s) 3.8 ± 0.2* 3.8 ± 0.2* 3.7 ± 0.1* 3.7 ± 0.2*
Sprint 5 (s) 3.8 ± 0.1* 3.8 ± 0.2* 3.8 ± 0.1* 3.8 ± 0.2*
Sprint 6 (s) 3.9 ± 0.2* 3.9 ± 0.2* 3.8 ± 0.1* 3.9 ± 0.2*
Sprint 7 (s) 3.9 ± 0.1* 3.9 ± 0.2* 3.9 ± 0.1* 3.9 ± 0.1*
Total sprint time (s) 26.1 ± 1.0 26.2 ± 1.0 26.0 ± 0.6 26.0 ± 1.0
20-m sprint 3.5 ± 0.1 3.5 ± 0.2 3.5 ± 0.1 3.4 ± 0.1

P < 0.0001 Effect of sprint, *indicates a significant difference from Sprint 1.

no group × time × sprint interaction effects for sprint times (P


= 0.96). There were no group, time or group × time interactions
for total time, maximum, mean and minimum power and fatigue
index during the RAST (all P > 0.05) (Figure 2). There was no
effect of group (P = 0.91), time (P = 0.50) or group × time
interaction (P = 0.25) for the 20-m sprint test.

DISCUSSION
This study aimed to investigate the effect of BA supplementation
in high-level Brazilian female soccer players during a three- FIGURE 2 | Sprint performance during the RAST for the beta-alanine (A) and
week preparatory training period. The main findings showed that placebo (B) groups pre- (clear bars) and post- (striped bars) supplementation.
*P < 0.0001. Effect of sprint number.
responses to BA supplementation were not different from those
obtained with placebo and were unable to avoid decreases in
performance during the YoYo IR1, which likely occurred due to
high workloads imposed in this preparatory period. There were
no changes in repeated or maximal sprint performance. athletes prior to the training and supplementation intervention
The training load employed with these athletes resulted in a was lower than that shown previously in elite female footballers
reduced exercise capacity during the YoYo IR1 (−7.4%). Prior to (7, 9), which provides support for this theory.
major events, such as an international tournament, it is common Short-term BA supplementation (3 weeks) was unable
to taper, namely reducing the training load from a previously to attenuate this training-associated decline in YoYo IR1
intense program in order to optimise gains and recovery and performance. The lack of an effect shown here is line with
maximise subsequent performance (14, 15). Our data suggest previous research showing no changes in YoYo IR1 in young elite
that, not only was the training intervention too intense in nature male basketball players with BA supplementation (20), although
to illicit improvements in exercise capacity, it actually worsened BA did improve YoYo IR2 (19) in amateur male footballers. The
performance which is contrary to the aims of the training. YoYo IR2 is initiated at a higher intensity than the YoYo IR1,
Previous data from an under-20 female football team preparing with a higher contribution from anaerobic glycolytic pathways
for the World Cup showed a progressive improvement in YoYo increasing the contribution of buffering capacity to performance
IR1 performance leading up to competition (Tunstall H, personal (9), making it more susceptible to improvements with BA. It is
communication in (6)), although the authors suggest this was also possible that the relatively short loading period in this study
reflective of a more focused fitness training schedule and the low did not meet the threshold necessary for a sufficient increase
starting fitness levels of these female players. In the current study, in muscle carnosine to elicit performance improvements. Smith
our athletes all plied their trade for top-level national sides, and et al. (28) showed similar improvement in cycling capacity
most were regular starters for their respective teams. It is possible following 3 weeks of high-intensity interval training with both
that a long grueling season took its toll on the players, and that BA and PL, but greater improvements from weeks 3–6 were
performance was a result of accumulated fatigue over the season shown with BA. It is currently unknown what the minimal
and would have reduced over this three-week period regardless necessary increase in muscle carnosine is to elicit a performance
of the intense training. In fact, YoYo IR1 performance of these improvement (29) and any definitive conclusions here are not

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Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers

possible due to the lack of muscle carnosine content analysis in PRACTICAL APPLICATIONS
the current study. Had the athletes commenced supplementation
prior to the training phase, thus ensuring increased muscle Intense preparatory training periods prior to international
carnosine content prior to the intense training period, it is competitions may place unnecessary strain on top-level
possible that results may have been different. However, we were footballers following a grueling season. Our data suggest
unable to enforce the supplementation protocol prior to the that international teams may inadvertently overload
international period during which we had access to the players, their players leading to a reduced high-intensity exercise
a potential consequence of working with elite club players on capacity in these players. Reduced exercise capacity was
international duty. As it stands, short-term BA supplementation not counteracted by short-duration BA supplementation,
was unable to attenuate the decline in YoYo IR1 performance although it is possible that the supplementation protocol
following the training period in this study. was sub-optimal. Perhaps more communication between
Neither training nor supplementation led to changes in clubs and international teams may facilitate this process and
repeated sprint ability or sprint performance. These data are avoid overreaching or overtraining, while prior initiation
in line with previous studies showing no effect of BA on of the supplementation protocol would further benefit any
short-duration repeated sprints in team sports players (21, potential adaptation.
22). However, previous research in female football players
has shown BA supplementation to improve mean power CONCLUSIONS
output during repeated 30-s Wingate sprints (30) and induce
greater improvements in repeated-sprint tests when combined Top-level female footballers involved in this intense 3-week
with plyometric training compared to training alone (11). training period prior to a competition worsened their high-
It is possible that the highly trained nature of our athletes intensity intermittent exercise capacity, and this negative result
contributed to these results, since meta-analytical data has was not attenuated by a short-duration BA supplementation
shown well-trained individuals to achieve smaller performance protocol throughout the same period. Further work is necessary
gains with supplementation than non-trained individuals (17). to elucidate whether adapted training protocols and BA dosing
The aforementioned studies recruited university level (30) and regimens could lead to better results.
amateur (11) players, while we employed elite youth players. It is
also important to again emphasise that the lack of any changes DATA AVAILABILITY STATEMENT
in these tests may similarly be due to the intense nature of the
training program, inhibiting any potential adaptations with or The datasets generated for this study are available on request to
without supplementation. the corresponding author.
One of the strengths of this study is that it was a real-
world intervention in which we implemented a double-blind ETHICS STATEMENT
placebo-controlled supplementation protocol in top-level female
athletes who were part of a competitive international set-up The studies involving human participants were reviewed and
performing their normal pre-competition training program. The approved by University of São Paulo. The patients/participants
standardized pre-tournament training camp provided a unique provided their written informed consent to participate in
environment that required all athletes to undergo identical daily this study.
routines such as training, nutritional intake and sleep, thus
removing several variables which could contribute to individual AUTHOR CONTRIBUTIONS
variability. Indeed, our data showed striking consistency with all
but one individual in BA showing a reduction in distance covered GR, AR, EP, VC, MB, PG, BG, and BS contributed to the
during the YoYo IR1, while six athletes in PL also covered less conception and design of the study. RR, EP, and BS organized the
distance; however, statistical analysis revealed that only two in BA database and performed the statistical analysis. RR, BD, AG, EP,
and one in PL could be considered to have worsened performance and BS wrote the first draft of the manuscript. GR, AR, VC, MB,
with >90% certainty (25). The controlled nature of this study has PG, and BG contributed to the subsequent versions. All authors
great practical applicability to similar athletes undergoing these contributed to manuscript revision, and read and approved the
types of intervention by showing BA to be ineffective during such final submitted version.
a short and intense training period. However, alternative methods
might be implemented, such as lighter training loads and earlier FUNDING
implementation of the supplementation regime, which might
lead to different results. Indeed, this study also highlights the No specific funding was received for this investigation. BD
delicate nature of working with an international team since we (2019/06140-5) and BS (2016/50438-0 and 2017/04973-4) have
could not make any changes to their usual routines until their been financially supported by Fundação de Amparo à Pesquisa
individual seasons with their respective national clubs had ended. do Estado de São Paulo.

Frontiers in Nutrition | www.frontiersin.org 5 April 2020 | Volume 7 | Article 43


Ribeiro et al. Short-Duration Beta-Alanine Supplementation Female Footballers

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65. doi: 10.1249/mss.0b013e31806010e0 for this and further experimental investigations and supported open access page
15. Mujika I. Intense training: the key to optimal performance charges for numerous publications involving the authors. NAI have not had any
before and during the taper. Scand J Med Sci Sports. (2010) input (financial, intellectual, or otherwise) into this original investigation.
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16. Harris RC, Tallon MJ, Dunnett M, Boobis L, Coakley J, Kim HJ, et al. The remaining authors declare that the research was conducted in the absence of
The absorption of orally supplied beta-alanine and its effect on muscle any commercial or financial relationships that could be construed as a potential
carnosine synthesis in human vastus lateralis. Amino Acids. (2006) 30:279– conflict of interest.
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17. Saunders B, Elliott-Sale K, Artioli GG, Swinton PA, Dolan E, Roschel H, et al. Copyright © 2020 Ribeiro, Duarte, Guedes da Silva, Ramos, Rossi Picanço, Penna,
Beta-alanine supplementation to improve exercise capacity and performance: Coswig, Barbalho, Gentil, Gualano and Saunders. This is an open-access article
a systematic review and meta-analysis. Br J Sports Med. (2017) 51:658– distributed under the terms of the Creative Commons Attribution License (CC BY).
69. doi: 10.1136/bjsports-2016-096396 The use, distribution or reproduction in other forums is permitted, provided the
18. Maughan RJ, Burke LM, Dvorak J, Larson-Meyer DE, Peeling P, original author(s) and the copyright owner(s) are credited and that the original
Phillips SM, et al. IOC consensus statement: dietary supplements publication in this journal is cited, in accordance with accepted academic practice.
and the high-performance athlete. Br J Sports Med. (2018) No use, distribution or reproduction is permitted which does not comply with these
52:439–55. doi: 10.1136/bjsports-2018-099027 terms.

Frontiers in Nutrition | www.frontiersin.org 6 April 2020 | Volume 7 | Article 43

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